| INVESTIGATION

Construction of Comprehensive Dosage-MatchingCore Histone Mutant Libraries for

Saccharomyces cerevisiaeShuangying Jiang,*,† Yan Liu,* Ann Wang,‡ Yiran Qin,* Maoguo Luo,* Qingyu Wu,* Jef D. Boeke,‡,1

and Junbiao Dai*,†,1

*MOE Key laboratory of Bioinformatics and Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University,Beijing 100084, PR China, †Center for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology,

Chinese Academy of Sciences, Shenzhen 518055, China, and ‡Institute for Systems Genetics and Department of Biochemistry andMolecular Pharmacology, New York University Langone Medical Center, New York, New York 10011

ORCID ID: 0000-0002-6786-6533 (S.J.)

ABSTRACT Saccharomyces cerevisiae contains two genes for each core histone, which are presented as pairs under the control of adivergent promoter, i.e., HHT1-HHF1, HHT2-HHF2, HTA1-HTB1 and HTA2-HTB2. HHT1-HHF1, and HHT2-HHF2 encode histone H3 andH4 with identical amino acid sequences but under the control of differently regulated promoters. Previous mutagenesis studies werecarried out by deleting one pair and mutating the other one. Here, we present the design and construction of three additional librariescovering HTA1-HTB1, HTA2-HTB2, and HHT1-HHF1 respectively. Together with the previously described library of HHT2-HHF2 mu-tants, a systematic and complete collection of mutants for each of the eight core S. cerevisiae histone genes becomes available. Eachdesigned mutant was incorporated into the genome, generating three more corresponding libraries of yeast strains. We demonstratedthat, although, under normal growth conditions, strains with single-copy integrated histone genes lacked phenotypes, in some growthconditions, growth deficiencies were observed. Specifically, we showed that addition of a second copy of the mutant histone genecould rescue the lethality in some previously known mutants that cannot survive with a single copy. This resource enables systematicstudies of function of each nucleosome residue in plasmid, single-copy, and double-copy integrated formats.

KEYWORDS histone H2A; histone H2B; histone H3; histone H4; mutagenesis

IN the genomes ofmost eukaryotes, there aremultiple genesencoding each histone protein, and these genes are often

distributed throughout the chromosomes (Maxson et al.1983; Marzluff et al. 2002, 2008), making it difficult, if notimpossible, to introduce a particular mutation into all copiesof a given histone gene simultaneously. Saccharomyces cere-visiae has only two copies of genes encoding each core histone,and these exist as pairs driven by bidirectional promoters, i.e.,HHT1-HHF1 and HHT2-HHF2 for histone H3 and H4, and

HTA1-HTB1 and HTA2-HTB2 for histone H2A and H2B(Hereford et al. 1979; Wallis et al. 1980; Choe et al. 1982;Smith and Andresson 1983; Smith and Murray 1983). Moreimportantly, the presence of either copy can support cell via-bility (Rykowski et al. 1981; Kolodrubetz et al. 1982; Smithand Stirling 1988; Dai et al. 2010), making the budding yeastan ideal system for high-throughput mutagenesis studies.Several mutant libraries have been constructed and used toprobe the function of histones (Hyland et al. 2005;Matsubaraet al. 2007; Dai et al. 2008, 2010; Nakanishi et al. 2008;Sakamoto et al. 2009; Govin et al. 2010; Choy et al. 2011;Sen et al. 2015; Luo et al. 2016).

The expression of histone genes is tightly regulated at boththeRNAandprotein levels during the cell cycle (Eriksson et al.2012). In S. cerevisiae, the two gene pairs for histone H3 andH4 are not expressed equally.HHT2-HHF2 contributes.80%of the H3–H4 mRNAs within a cell (Cross and Smith 1988).However, in the absence of HHT2-HHF2, transcription of

Copyright © 2017 by the Genetics Society of Americadoi: https://doi.org/10.1534/genetics.117.300450Manuscript received May 24, 2017; accepted for publication October 20, 2017Supplemental material is available online at www.genetics.org/lookup/suppl/doi:10.1534/genetics.117.300450/-/DC1.1Corresponding authors: Institute for Systems Genetics and Department ofBiochemistry and Molecular Pharmacology, New York University LangoneMedical Center, New York, NY 10011. E-mail: jef.boeke@nyumc.org; and MOEKey laboratory of Bioinformatics and Center for Synthetic and Systems Biology,School of Life Sciences, Tsinghua University, 2-305 Biotech Bldg., Beijing 100084,PR China. E-mail: jbdai@tsinghua.edu.cn

Genetics, Vol. 207, 1263–1273 December 2017 1263

HHT1-HHF1 can be upregulated to support cell growth,although minor defects in chromosome segregation andDNA repair have been observed in such strains (Smith andStirling 1988; Liang et al. 2012). Although knocking outHHT1-HHF1 results in no obvious phenotypic flaw as tested(Cross and Smith 1988), previous work has shown that cer-tain histone mutants, driven by the native promoter, werelethal when integrated at HHT2-HHF2 locus, even thoughthey were viable when introduced as a CEN plasmid, presum-ably due to increased plasmid copy number (Matsubara et al.2007; Dai et al. 2008; Nakanishi et al. 2008; Sakamoto et al.2009). These observations suggest that such mutants arehypomorphs that can be phenotypically rescued by increasinggene dosage. These observations also suggested that complexand difficult to interpret phenotypes could be introducedduring mutagenesis studies using libraries carrying only asingle copy of histone genes. Specifically, such phenotypecould result from some unknown combination of the muta-tion and the reduced gene dosage.

Here, we present the design and construction of three newlibraries of histone mutants, two covering H2A/H2B and theother H3/H4, that both complete the set of all four corehistones, and also can be used to restore gene dosage tothe native state. As in the original H3/H4 library, we system-atically changed each residue to alanine, and replaced alaninewith serine. When possible, each modifiable residue wassubstituted with amino acids mimicking both modified andunmodified states, allowing exploration ofmodification state.Additional mutations were introduced to either eliminate, oreven to reverse side chain charge and size. Furthermore,systematic deletion mutants at both N- and C-termini wereincluded, generating a comprehensive library of histone H2Aand H2B mutants with 592 alleles. The first library allowsintegration of each mutant at HTA1-HTB1 locus, and the sec-ond library targets the HTA2-HTB2 locus. Based on the pre-vious library design, we constructed a complementary libraryallowing eachmutant to be separately integrated at theHHT1-HHF1 locus. All three libraries are provided as bacterial stocksto allow user-defined utilization. Yeast strains with either asingle copy, or two copies of the same mutants integrated atthe native histone loci, were constructed and provided.

Materials and Methods

Construction of bacterial libraries of 592 H2A/H2B mutants

The mutant constructs (H2ML1, driven by the HTA1/HTB1promoter) were synthesized and cloned into pRS416 by Ep-och Life Science (http://epochlifescience.com/). The plas-mids were supplied as bacterial stocks in a 96-well format.To construct the second copy of mutants (H2ML2, driven bythe HTA2/HTB2 promoter), plasmids were isolated, dilutedby 50-fold, and used as template to amplify mutated frag-ments. The purified digested PCR products were then clonedinto destination plasmids pJD411 (H2A mutants, BglII/XhoI)

and pJD412 (H2B mutants, ClaI/SalI) respectively. Plasmidswere isolated, and sequence verified to ensure 100% accu-racy. These plasmids were arrayed and stored as bacteriastocks in the same order as that of H2ML1.

Construction of the second bacterial library of 562 H3/H4 mutants

Seventy-six point mutants with charge reversal of H3 and H4were newly designed and constructed from the WT base con-structs (pJD47 forH3 andpJD62 forH4) to expand the previousH3/H4 library (Table 1) (Dai et al. 2008), generating theupdated H3/H4 library with 562 alleles (H3/4ML2, Table 2).

To construct the second copy of H3/H4 mutants (H3/4ML1, driven by the HHT1/HHF1 promoter), the previouslymade plasmids (Dai et al. 2008) were isolated and diluted50-fold as above. The purified digested PCR products werethen cloned into chloramphenicol-resistant destination plas-mids pJD233 (H3 mutants, SalI/ClaI) and pJD232 (H4 mu-tants, BglII/SphI) respectively. Plasmids were isolated, andsequence verified to ensure 100% accuracy. These plasmidswere arrayed and stored as bacteria stocks in the same orderas that of H3/4ML2.

Construction of yeast libraries of histone mutants

To construct the yeast H2A/H2B library, each plasmid(H2ML1) was digested by BciVI and NcoI (located withinthe ORF of URA3) to release the mutant constructs beforethey were transformed into the host strain JDY142. Correctintegration was confirmed by colony PCR. PCR confirmedcells were plated onto medium containing 5-fluoroorotic acid(5-FOA) to eliminate pJD78 (HTA2-HTB2 in pRS316), theplasmid containing wild-type histone H2A and H2B genes.Viable strains were used to integrate the second copy of thesame mutants (H2ML2). A BciVI digestion was done beforetransformations of the H2ML2 mutants.

To construct yeast strainswith double-copy integratedH3/H4mutants, viable strains with single-copymutants in S288Cbackground (Dai et al. 2008), and updated in this study, wereused for the integration of BciVI digested mutant fragmentsof H3/4ML1.

Growth competition assay

Three independent colonies of each strain were inoculatedinto fresh YPD medium, cultured at 30� overnight, and thensubcultured for an additional 5 hr from a starting A600 of�0.1. The cell density was determined and the cell cultures(single-copy with mCherry at the HO locus) were mixed at a1:1 ratio. Mixed cells were inoculated into fresh YPDmediumwith a starting cell concentration of �53 105 cells/ml. Theywere cultured at 30� and diluted to 5 3 105 cells/ml every12 hr for a total of 36 hr. Cells were collected and resus-pended in PBS buffer with 0.1% Tween as FACS samples ateach transfer time point and final time point. An LSR Fortessacytometer (BD Biosciences) with a high throughput sampler(HTS) loader was used to determine the total cell numberand the proportion of mCherry positive cells by analyzing

1264 S. Jiang et al.

50ml cell suspension for each sample. The fitness was calcu-lated as described (Thompson et al. 2006).

Chromatin fractionation assay

The chromatin fractionation assay was conducted following aprevious paper (Liang and Stillman 1997). In brief, 50 ml cellsat �8 3 106 cells/ml were harvested and resuspended inprespheroplasting buffer. After incubation for 10 min, cellswere digested with zymolyase 100T (120493; Amsbio) atroom temperature on a rotator. Then, spheroplasts werewashed with ice-chilled wash buffer, and resuspended in anequal pellet volume of extraction buffer (EB). Spheroplastswere lysed by adding 1/40 vol 10% Triton X-100 (final con-centration at 0.25%), and incubated on ice for 3 min withgentle mixing; the resulting sample was defined as the wholecell extract (WCE). Lysate was underlayered with 50% vol of30% sucrose and spun at 12,000 rpm for 12 min. The upperyellow layerwas the supernatant fraction. Chromatin pelletwaswashed and resuspended again with EBX (EB+0.25%TritonX-100). The samples of the whole cell extract, supernatant,and pellet were boiled and subjected to immunoblotting.

Data availability

The base yeast strains are available upon request, and will bedeposited to the American Type Culture Collection (ATCC).

The libraries will be deposited to Addgene as plasmids. Sup-plemental Material, File S1 contains detailed descriptions ofall supplemental data, including seven supplemental figuresand four supplemental tables.

Results and Discussion

Design of 592 H2A/H2B mutants

All currently available mutant libraries of histone H2A andH2B can only be used in an episomal plasmid format, whichpotentially limits their application in some situations, partic-ularlywhen a single gene copy is strictly required. In addition,despite the fact that multiple libraries are available, themutations are mostly limited to alanine substitutions or afew modifiable residues. A comprehensive library of histoneH2A and H2B mutants would help further dissection ofnucleosome function. Therefore, we designed a new collec-tion of histoneH2AandH2Bmutants, systematically coveringevery residue in the two core histones (Table 1).

The mutants could be divided into three types (Table 1):(1) Point mutants. We substituted each amino acid residuewith alanine and changed native alanine to serine, allowingus to probe the function of the side chain of every residue.To study the influence of modifications, we systematically

Table 1 The mutagenesis strategy of the histone libraries

Category Original Residue New Residue Rationale H2A (No.) H2B (No.) H3 (No.) H4 (No.)

Point mutant All Alanine(A) Remove sidechain 112 113 119 96H2A/H2B: 516 Alanine(A) Serine(S) Alter sidechain 19 17 16 6H3/H4: 477 Lysine(K) Arginine(R) Mimic deacetyl 11 19 16 11

Lysine(K) Glutamine(Q) Mimic acetyl 11 19 16 11Arginine(R) Lysine(K) Mimic demethyl 10 6 17 14Serine(S) Aspartic Acid(D) Mimic phosphate 8 19 10 6Threonine(T) Aspartic Acid(D) Mimic phosphate 5 11 9 6Tyrosine(Y) Glutamic Acid (E) Mimic phosphate 3 5 2 4Lysine(K) Glutamic acid(E) Reverse charge 11 19 16 11Arginine(R) Glutamic acid(E) Reverse charge 10 6 17 14Aspartic Acid(D) Arginine(R) Reverse charge 3 3 4 3Glutamic Acid(E) Arginine(R) Reverse charge 5 8 7 4Histidine(H) Glutamine(Q) Remove charge 3 2 2 2Aspartic Acid(D) Asparagine(N) Remove charge 3 3 4 3Glutamic Acid(E) Glutamine(Q) Remove charge 5 8 7 4Asparagine(N) Aspartic Acid(D) Add charge 8 3 1 1Glutamine(Q) Glutamic Acid(E) Add charge 6 4 8 2Tyrosine(Y) Phenylalanine(F) Remove OH 3 5 2 4Proline(P) Valine(V) Block

isomerization5 5 2 –

Deletion mutant N Tail deletion —— —— 10 45 52 27H2A/H2B: 68 C Tail deletion —— —— 10 3 – –

H3/H4: 79Comprehensive mutant Multiple Tail

Lysines(K)Alanine(A)/

Glutamine(Q)/Arginine(R)

Mimic unmodi-fied/acetyl/deacetyl

3 3 3 3

H2A/H2B: 8 Compound sub-stitutions

—— Mimic WTHta1p/Htb1p

1 1 – –

H3/H4: 6Total 265 327 330 232

H2A/H2B: 592H3/H4: 562

Bold font indicates new mutagenesis strategies, which were not included in the previous histone H3/H4 library (Dai et al. 2008).

Libraries of Histone Mutants in Yeast 1265

changed any modifiable residues to mimic either modified orunmodified form. Additionally, and differently from the firstlibrary we described (Dai et al. 2008), we deliberately swap-ped the charge status for a charged residue.We replaced eachlysine and arginine with a glutamate residue. Similarly, wereplaced each glutamate and aspartate with arginine. To-gether, these strategies generated 516 point mutants; (2) De-letion mutants. The N- and C-termini (“tails”) of histone H2Aand H2B are known to play critical roles in nucleosome func-tion. For example, serine 128 on histone H2A is known to bephosphorylated, and phosphorylation is required to recruitimportant protein factors during the process of repairingdamaged DNA (Downs et al. 2000; Morrison et al. 2004). Atotal of 68 systematic deletion mutants was designed toremove sets of four residues at either N- or C-terminal of his-tone H2A and H2B; (3) Multi-point mutants. Since multiplelysines are modified within the N-termini of histones H2A andH2B, we constructed six mutants that allowed us to replacemultiple lysines with alanine, arginine or glutamine at eachposition. Therefore, potential modifications for all of theseresidues could be removed or mimicked simultaneously. Inaddition, since the two copies of genes encode slightly differ-ent amino acid sequences for both histone H2A and H2B, andthemajority of themutants were generated based on theHTA2and HTB2 amino acid sequences, specific substitutions weremade to convert the amino acid sequences to matchHTA1 andHTB1. Eventually, we designed a total of 592 alleles, including265 mutants in histone H2A and 327 mutants in histone H2B(Table 1), producing the most comprehensive histone H2Aand H2B mutant library available.

We generated each mutant based on the amino acid se-quences of HTA2 and HTB2 (Figure S1A in File S1), whichwere codon-optimized and recoded synonymously usingGeneDesign (Richardson et al. 2006) to avoid potential ho-mologous recombination between the synthetic constructand the native gene. Since HTA1-HTB1 in the genome suf-ficed for normal growth while HTA2-HTB2 did not (Norrisand Osley 1987; Moran et al. 1990; Libuda and Winston2006), the former locus was chosen for the integration forthe synthetic cassette. To keep the expression of the histonesH2A/H2B similar to that of the native form, the endogenousHTA1-HTB1 promoter was used to control expression. Weswapped the native terminators of both genes with eitherCYC1 or ADH1 terminator, to eliminate recombination withwild-type sequences, which could potentially lead to inad-vertent swapping of the histone mutant with the wild-type

sequence. The synthetic histone cassette was flanked by na-tive sequences upstream and downstream of the HTA1-HTB1locus, allowing the construct to be integrated at this locus,replacing any DNA inbetween. For selection, a LEU2 genewas inserted between one homologous region and the 39end of the UTR sequences adjacent to the histone codingsequence that was mutated. Thus, the set of H2A mutantswas made in one parental plasmid, and the set of H2B mu-tants was made in a separate one. The overall construct de-sign is shown in Figure 1A.

Importantly, eachmutantwasmarkedwith apair of uniqueDNA sequences (molecular barcodes or TAGs), taking from asubset of the molecular barcodes used in the yeast knockoutcollection (Winzeler et al. 1999). Each barcodewas flanked by apair of universal primers and placed adjacent to each other be-tween the 39UTRof the histonemutant and the LEU2 gene. Thepresence of the TAGs allows pooling of themutants and analysisof some otherwise very labor-intensive assays, such as thosedemonstrated using the yeast knockout collection (Smith et al.2009, 2010, 2012; Gresham et al. 2011; Gibney et al. 2013).

The synthetic cassette was cloned into a bacteria-yeastshuttle vector pRS416, allowing propagation and amplifica-tion of each mutant in Escherichia coli. At the same time, eachconstruct could be transformed directly into a yeast strainand used as a replication-competent episome. The syntheticcassette can also be released after a single restriction enzymedigestion, and transformed into the host strain to integrate atthe native locus with high fidelity. In addition, to further in-crease the utility of this library, the LEU2 genewas flanked by apair of loxP sites, allowing the LEU2 gene to be either removedor “swapped” with other selectable markers, as needed.

A second histone H2A and H2B mutant library targetingthe HTA2-HTB2 locus

As described above, the initial library of histoneH2A andH2Bwas under the control of HTA1-HTB1 promoter, and config-ured to integrate at HTA1-HTB1. Since the two pairs of his-tone genes are regulated differently in yeast (Norris andOsley 1987; Moran et al. 1990; Libuda and Winston 2006),we reasoned that we could minimize the impact of gene dos-age on phenotype by integrating two copies of the same mu-tant at their two native loci, each under control of itsendogenous promoter. Therefore, a second mutant librarywas designed and constructed (Figure 1B).

To construct this library, the base constructs containingwild-type histone H2A and H2B were generated by replacing

Table 2 Formats of the histone mutant libraries (plasmids)

Name Histone

CENFormatMarker

IntegratedMarker

IntegratedLocus

E. coliSelectiveMarker

No. ofMutants

Publishedor not

H2ML1 H2A/B URA3 LEU2 HTA1-HTB1 AmpR 592 This studyH2ML2 H2A/B TRP1 NatMX4 HTA2-HTB2 CmR 592 This studyH3/4ML1 H3/4 TRP1 HygMX4 HHT1-HHF1 CmR 562 This studyH3/4ML2 H3/4 TRP1 URA3 HHT2-HHF2 AmpR 562 Dai et al. (2008)

and this study

1266 S. Jiang et al.

theHTA1-HTB1 promoter withHTA2-HTB2 promoter, and bysubstituting the homologous sequences flanking HTA1-HTB1by sequences flanking the HTA2-HTB2. We used NatMX4(Goldstein and McCusker 1999) as a selection marker toidentify the integrants, enabling selection of strains contain-ing both copies of the mutants. We cloned this second syn-thetic cassette into centromeric plasmid pBC414 tagged withthe TRP1 marker (Frazer and O’Keefe 2007), allowing eachmutant to be supplied individually as an episome. The mu-tant histone sequences (with their TAGs) were amplified byPCR and cloned into the corresponding base construct (Ma-terials and Methods), generating the second library of histoneH2A and H2B mutants (H2ML2, Table 2).

A new library of histone H3 and H4 mutants

Previously, we reported the design of a versatile histone H3and H4 mutant library under the control of the HHT2-HHF2promoter, which could be used similarly to the H2A and H2Blibrary described above (Figure 1C) (Dai et al. 2008). Here,we first expanded the existing H3/H4 library to include the

charge reversal type mutants that were not included inthe original version (H3/4ML2, Table 1 and Table 2). Sincethe two histone H3/H4 loci are also regulated differently, wetherefore decided to construct a second library of mutants asrationalized above for histone H2A/H2B.

In the daughter library, the expression of histonesH3/H4 iscontrolled by the native HHT1-HHF1 promoter (Figure 1D).Sequences flanking HHT1-HHF1 were used as homologyarms. The selectable marker for integration, URA3 wasreplaced by HygMX4, and the synthetic cassette was alsocloned into pBC414 (Frazer and O’Keefe 2007). The mutanthistone sequences were amplified and cloned into the corre-sponding base construct (Materials and Methods). These ma-nipulations produced a new library of H3/H4 mutants,comprising 562 alleles (H3/4ML1, Table 2).

Four yeast libraries of viable histone mutants

For functional studies,we integrated eachhistonemutant intothe chromosome to generate a collection of yeast libraries. Tohost the histone H2A and H2B mutants, aMATa strain in the

Figure 1 (A–D) Versatile dosage match-ing libraries for all core histones. The li-braries (H2ML1, H2ML2, and H3/4ML1)marked in gray were newly generated inthis study; H3/4ML2 was updated fromthe H3/4 library reported previously (Daiet al. 2008). All libraries can be used asepisomal plasmids or integrated into thegenome after BciVI (marked with blue tri-angle) digestion. The synthetic constructsfor H2A, H2B, H3, and H4 mutants aredenoted as H2AS, H2BS, H3S, and H4S,respectively. The selectable markers (LEU2,URA3, NatMX4, and HygMX4) for integra-tion are always positioned adjacent to thegene bearing the mutation(s) to preventrecombination events between the markerand the mutant (i.e., within the promoterfragment). The synthetic histone geneswere driven by native promoters. The se-quences flanking histone ORFs were usedas homology arms. There are very minorcoding sequence differences betweenHTA1/B1 and HTA2/B2; for H2ML1 andH2ML2, wild type Hta2-Htb2 amino acidswere used as the reference sequence forrecoding to HTAS and HTBS. Each mutantwas labeled with unique TAG sequences(marked as yellow squares). The TAGscan be used for the identification of spe-cific mutants using high-throughput se-quencing. As in H3/4ML2, each plasmidin H2ML1 was designed with flankingloxP sites (red triangles) allowing facile“swap out” of the marker genes.

Libraries of Histone Mutants in Yeast 1267

S288C background was constructed by knocking out the ge-nomic HTA1-HTB1 and HTA2-HTB2 loci and supplied a wild-type HTA2-HTB2 on a URA3 CEN “shuffle” plasmid. All592 mutations were integrated at HTA1-HTB1 by selectingfor LEU2 and screening for loss of the resident KanMX4marker, and subsequently confirmed by PCR. The wild-typeplasmid was then removed either spontaneously by mitoticplasmid loss, or intentionally by counter-selection in mediumcontaining 5-FOA. The viable strains lacking the wild-typeplasmid constitute the yeast library with single-copy inte-grated H2A and H2B mutants (BY-H2ML1, Table 3). Oncethe above strains were constructed (BY-H2ML1, Table 3), westarted to incorporate the second copy of the histone mutant(H2ML2) into the HTA2-HTB2 locus of strains that alreadyhave the same mutant integrated at the HTA1-HTB1. Thestrains were selected in medium containing nourseothricin,and subsequently replicated ontomedium containing hygrom-ycin B to select for strains with correct replacement of markergenes. The candidates were further confirmed by PCR to en-sure that each mutant was positioned correctly. This producedthe yeast library of H2A/H2B with two doses of each mutantunder their native promoters (BY-H2ML1&2).

The viable H3/H4 mutant strains incorporated at theHHT2-HHF2 locus in S288C background (BY-H3/4ML2, Ta-ble 3) were used for subsequent integration of the newly builthistone H3/H4 mutant constructs. A similar series of stepswas used to produce the new library of histone H3/H4 withtwo doses of each mutant (BY-H3/4ML1&2, Table 3).

Thus, a total of four yeast libraries now exists, in whichthere is either one or two doses of the same mutant in eachstrain. These libraries represent a valuable resource to studythe influence of specific histone residues, tails, and surfaces onnucleosome function.

Histone dosage defect produces phenotypes

In our previous work on histones H3 and H4, we identified27 lethal mutants in which original residues were mutated toalanine in the S288C background (Dai et al. 2008). However,another library, in which the expression of H3/H4 was drivenby the same promoter on CEN plasmids, identified only 14 le-thal mutants (Nakanishi et al. 2008). One possible reason forthe significant difference between two studies is the dosage

of histone genes. Therefore, three histone H3 mutants (I62A,L103A, and L126A), reportedly lethal when integrated butviable as episomal plasmid, were randomly chosen andtested. Consistently, all three mutants failed to propagate inthe single copy integrated format. In contrast, incorporating asecond copy completely rescued lethality (Figure 2A). Addi-tionally, H3 F84A showed an obvious growth defect with onlya single copy, which could be rescued when there are twocopies of the mutant genes. These observations strongly sup-port that it is important to retain both histone copies forfunctional analyses, and maintain the native regulation ofhistone gene expression (Eriksson et al. 2012) as well as genedosage. To better understand the influence of histone copynumber on phenotypes, the growth of wild type strains con-taining different copy numbers of synthetic H2A/H2B geneswere compared in rich medium. We found no obvious differ-ence in YPD medium by either a growth competition assay(Figure 2B) or serial dilution (Figure 2C and Figure S1B inFile S1). The expression levels of histone H2A also did notshow significant differences among strains, both in protein andmRNA level (Figure 2D and Figure S2 in File S1). This suggeststhat the proteins expressed by single-copy histone genesdriven by the promoter of HTA1-HTB1 in wild-type strainsare sufficient to support normal growth, consistent with pre-vious studies (Norris and Osley 1987; Moran et al. 1990).

Next, strains with either one or two copies of wild typeH2A/B genes were tested for sensitivity to a series of stresses.All strains showed no growth difference under most condi-tions tested (Figure S1B in File S1). However, on plates con-taining benomyl, strains with single-copy histones showedslower growth than the control strain BY4742, and strainswith double-copy histones (Figure 2C and Figure S1B in FileS1). It suggests that the copy number of histone genes doesaffect phenotypes of strains, and that strains with two copiesof synthetic H2A/H2B behave more like the wild-type strain.We then carried out a parallel high-throughput test usingstrains containing either a single copy of a histone mutant,or two copies of the same mutant upon treatment with beno-myl, hydroxyurea (HU), and methyl methanesulfonate(MMS). Substantial phenotypic variations were detected.Some mutants only showed sensitivity to a drug in onestrain, and some displayed differential sensitivity (Table

Table 3 Yeast libraries (S288C background) of the histone mutants

Format Name Mating TypeGenotype forthe Strains Published or not

Single-copy BY-H2ML1 a his3D200 leu2D0 lys2D0 trp1D63 ura3D0 met15D0 hta2-htb2::HygMX4 hta1-htb1::LEU2-HTAS-HTBS

This study

BY-H3/4ML2 a his3D200 leu2D0 lys2D0 trp1D63 ura3D0 met15D0 can1::MFA1pr-HIS3 hht1-hhf1::NatMX4 hht2-hhf2::URA3-HHTS-HHFS

Dai et al. (2008) andthis study

Double-copy BY-H2ML1&2 a his3D200 leu2D0 lys2D0 trp1D63 ura3D0 met15D0 hta2-htb2::NatMX4-HTAS-HTBS hta1-htb1::LEU2-HTAS-HTBS

This study

BY-H3/4ML1&2 a his3D200 leu2D0 lys2D0 trp1D63 ura3D0 met15D0 can1::MFA1pr-HIS3 hht1-hhf1::HygMX4-HHTS-HHFS hht2-hhf2::URA3-HHTS-HHFS

This study

1268 S. Jiang et al.

S1 in File S1). Several mutants were randomly selected toconfirm the high-throughput results (Figure 2E and FigureS4 in File S1). For H2B S67D, strains with double-copyH2A/H2B were, paradoxically, more sensitive to MMS thanthose with single-copy histones. In contrast, strains withH2A Y58F showed the opposite sensitivity pattern to beno-myl. These results indicate that the copy number of histonemutants may contribute significantly to their phenotypes,and the effect is complex, depending on the mutation. Themutant library with two copies of the same mutant histone

under the native promoters seems like the best strategyfor minimizing potential artifacts related to gene dosage.

In budding yeast, the two copies of histone H2A and H2Bgenes are nonidentical in terms of protein sequence (FigureS1A in File S1). In the library, the amino acid sequences ofHta2p and Htb2p were used as the reference to recode theDNA sequence. One potential drawback for this design is thatit was possible that amino acid differences between the twocopies of histone might themselves influence strain fitness.Therefore, we systematically assayed phenotypes of strains

Figure 2 Histone dosage defect produces phenotypes. (A) Viability test of histone mutants by plasmid shuffling. In the listed strains, histone mutantswere integrated into HHT2-HHF2 site or both loci and wild-type H3/H4 were expressed from a CEN-URA3 plasmid. Cells were cultured at 30� for 3 days.(B) No significant growth advantage in strains with double-copy vs. single-copy histone genes. mCherry was integrated at the HO site in strains withsingle-copy histones to quantify the composition of cell populations. Cultures were inoculated by mixing equal numbers of cells (2.5 3 105 cells/ml foreach type). The cells were cocultured in YPD medium at 30� and diluted to 5 3 105 cells/ml every 12 hr for a total of 36 hr. Cells were collected atcorresponding time points and then subjected to flow cytometry. H2AS WT, strains with synthetic H2A base construct; H2BS WT, strains with syntheticH2B base construct. D, strains with double-copy integrated histones; S, strains with single-copy integrated histones. Data were represented as mean 6SD (C) Strains with single-copy histones were more sensitive to benomyl than those with double-copy histones (BY4742 and H2AS/H2BS WT D). Cellswere diluted fourfold and spotted onto YPD or YPD with benomyl. (D) The total expression of histone H2A was measured by immunoblotting in strainsat different histone dosages. (E) H2B mutants with different histone dosage showed differential sensitivity to MMS/benomyl. Cells were diluted by10-fold, and spotted onto YPD or YPD with drugs.

Libraries of Histone Mutants in Yeast 1269

containing the two versions of the H2A or H2B sequences.Wefound no difference among these strains, no matter whetherthey were present in one or two copies (Figure S1B in File

S1). These data demonstrate that HTA1-HTB1 and HTA2-HTB2 function equivalently when used as the sole histoneH2A and H2B source.

Figure 3 Altering charged residues severely affects viability of histone mutants. (A) Comparison of lethal mutants between previous screens (Matsubaraet al. 2007; Nakanishi et al. 2008) and results of this study. (B) Immunoblot analysis of total H2A and H2B expression in strains containing two copies oflethal histone mutants and Flag-His tagged wild-type histone plasmids. Tubulin was used as the loading control. (C) Chromatin fractionation to analyzeincorporation of histone mutants (H2B Y40E, S61D) into chromatin. The same strains as in (B) were used. CP, chromatin pellets; S, supernatant; W,whole cell extracts. Histone H3 and Pgk1p were used as indicators of chromatin and cytoplasm components. 40 A600 units of cells were harvested andused for each strain. (D) Evolutionary conservation scores for viable mutants and lethal mutants were calculated using ConSurf. **** P , 0.0001. Datawere represented as Mean 6 SEM (E) Classification of lethal mutants based on alternation of charge state. Pink, negative charge was converted topositive charge; blue, negative charge was removed; yellow, negative charge was added; green, positive charge was swapped by negative charge;orange, other point mutation not affecting charge; gray, lethal tail deletion mutants. (F) Lethal mutants, except several buried ones and tail deletions, aremarked in the nucleosome structure with the same colors used in (E). The PDB number of the nucleosome structure used in this paper is 1ID3 (yeast corenucleosome). (G) Viability test of histone mutants by plasmid shuffling. In the listed strains, histone mutants were expressed from a CEN-TRP1 plasmid,and wild-type HTA2-HTB2 were expressed from a CEN-URA3 plasmid.

1270 S. Jiang et al.

Lethality profiles of H2A and H2B mutants

Previously, five alanine substitution mutants were defined aslethal including H2A Y58A, E62A, R82A, D91A, and H2BL109A (Matsubara et al. 2007), with one (H2A R82A) ques-tionable (Nakanishi et al. 2008). Using strains containing twocopies of histone mutants and a wild type HTA2-HTB2 on acentromeric plasmid, we defined a mutant as “lethal” if itfailed to grow onmedium containing 5-FOA at 30� for 3 daysafter plating. Among the 592 mutants, 22 mutants in totalfailed to support cell viability (Figure 3A and Table 4), in-cluding the four known lethal mutants. In agreement withNakanishi’s work, we found H2A R82A to be viable in ourlibrary in both the integrated and episomal formats (FigureS5 in File S1). It is notable that the number of lethal mutantsin H2A/H2B library is substantially lower than in the H3/4library (22/592 of H2A/B mutants vs. 80/562 of H3/4 mu-tants, Table 4) (Dai et al. 2008). One possible explanation isthat, overall, the function of H2A and H2B is somewhat lesscritical compared to that of H3 andH4. This is consistent withthe fact that the bulk of H3 and H4 proteins occupy the upperhalf of the nucleosome where the DNA entry and exit pointsare, and also with the fact that the H2A/H2B amino acidsequences are less conserved. In addition, unlike H3/H4 mu-tants, the copy number of histone genes did not show signif-icant effects on the viability of H2A/H2B mutants, implyingthat cells need more tightly regulated expression of histoneH3 and H4 than that of H2A and H2B.

To rule out the possibility that a mutant is lethal because itfailed to be expressed in the cell, we carried out immunoblotexperiments in strains containing wild-type histones withN-terminal Flag-his tag expressed from a CEN plasmid. Asshown in Figure 3B, all mutated histone proteins were de-tectable, suggesting that none of them has transcriptionaland translational defects. One interesting observation is that,in the wild-type strains, the tagged native histones are sub-stantially underexpressed relative to that of untagged ones,whereas in the majority of the mutants, the abundance of thetagged native protein is significantly increased (Figure 3B).One possibility is that themutated histones are less efficientlyincorporated into chromatin, leading to the over accumula-tion of wild-type histones and subsequent degradation ofthe unincorporated mutant histones (Gunjan and Verreault

2003). To test this hypothesis, chromatin fractionation wasperformed in two randomly selected lethal histone H2B mu-tants: Y40E and S61D. As shown in Figure 3C, the percentageof histones from the synthetic construct incorporated into thechromatin showed similar proportions to that in totalexpressed histones, with more of the tagged wild-type his-tones in the chromatin in both mutant strains. This resultsuggests that the mutant histones are less preferable as chro-matin components than the wild-type histones, consistentwith the above hypothesis. Further analysis confirmed thatthe lethal mutants were generally more conserved aminoacid residues (Figure 3D).

Intriguingly, .85% (19 out of 22) of the lethal mutantsfell in residues with charge alterations (Figure 3E and Table4), which is a higher proportion than that of H3/4 lethalmutants (45/80, Table 4) (Dai et al. 2008). And 11 of thoselethal mutants came from the new charge reversal strategy(Table 4). When we mapped the position of these mutants tonucleosome structure, several features were revealed. (1) Allthree alleles swapping from positive to negative residues(K76E, R78E, and R30E) were positioned at surfaces thatcontact DNA (green, Figure 3F). The negative charge proba-bly affects the nucleosome structure by repelling the DNA. Insupport of this, we found that neutralizing the charges withalanine substitution would not impair cellular viability at allthree positions. (2) Several lethal mutant residues, such asH2A E57, E62, E65, D91, and E93, clustered at the “acidicpatch,” which interacts with positively charged residueswithin the histone H4 N terminal and is reportedly crucialfor formation of higher order nucleosome arrays (Luger et al.1997; Dorigo et al. 2004; Zhou et al. 2007; Song et al. 2014).Lethal mutations in these residues, such as E/D to R (in pink,Figure 3F) may cause lethality by compromising this impor-tant interaction. Remarkably, every mutation tested at posi-tions E62 (A/Q/R) and D91 (A/N/R) on histone H2A causeslethality, highlighting that these two positions are so criticalthat a wide variety of perturbation types cannot be tolerated.Since all mutants changed the charge status at both locations,we hypothesized that the negative charge might be pivotal.To test this hypothesis, additional mutants E62D and D91E,which contain slightly altered side chains but similar negativecharges, were constructed. As expected, H2A E62D was able

Table 4 Lethal mutants in the histone libraries

Category H2A H2B H2A&H2B H3a H4a H3&H4a

Lethal mutants 15 (5.7%) 7 (2.1%) 22 (3.7%) 44 (13.3%) 36 (15.5%) 80 (14.2%)Lethal point mutations 14 7 21 41 28 69Lethal residues 9 7 16 32 20 52Lethal mutants with

changed charge13 6 19 25 20 45

Lethal mutants withcharge reversal

8 3 11 11 9 20

Mutants with charge reversal 29 36 65 44 32 76Point mutations 241 275 516 275 202 477Total mutants 265 327 592 330 232 562a Indicates the results from both published data (Dai et al. 2008) and this study (Table S2 in File S1).

Libraries of Histone Mutants in Yeast 1271

to support cell viability (Figure 3G, upper panel). On theother hand, H2A D91E was lethal, indicating that charge isnot the sole determinant of lethality at this particular posi-tion. For other mutants described in groups 1 and 2 above, itis notable that the charge neutralization mutants at the sameresidues are viable. Therefore, we conclude that active re-pulsion of charge is substantially more disruptive than simplecharge neutralization. (3) In addition, some lethal mutantswere scattered among histone–histone interaction surfaces(Figure 3F and Figure S6 in File S1). These mutants mightaffect nucleosome structure integrity or chromatin assemblyby disrupting histone–histone interactions. (4) We previouslyidentified several N-terminal tail deletions in histones H3 andH4 as lethal. However, among all of the deletionmutants, onlyone of them (H2A del118-127) was lethal (Figure 3B), indi-cating that the tails on H2A and H2B are almost dispensable.

Compared to the lethal mutants in histone H3 and H4, thedistribution of histone H2A andH2B lethal mutants was quitedifferent. Most of the H2A/H2B mutants were located at thenucleosome surface (such as the acid patch, Figure 3F andFigure S7 in File S1) whereas many H3/H4 lethal mutantswere buried in the nucleosome core, besides those mutantstracking the DNA-binding surface (Figure S7, Table S2 in FileS1, and Table 4) (Dai et al. 2008). Additionally, the buriedlethal residues of H3/H4 are mostly located at the H3/H4interface, or the H3/H3 interface involved in the formation ofH3/H4 tetramers, rather than the interface with H2A or H2B.The above features are reminiscent of the spatial and tempo-ral order of nucleosome assembly in which H3/H4 tetramer isdeposited to DNA first and H2A/H2B heterodimers are sub-sequently loaded to complete the core particles (Worcel et al.1978; MacAlpine and Almouzni 2013). Therefore, the lethal-ity of histoneH3/H4mutants aremore likely due to defects inlocal nucleosome structure or assembly, whereas H2A/H2Blethal mutants might interfere more with the interactionsbetween nucleosomes and other protein factors or adjacentnucleosomes.

Acknowledgments

We are grateful for financial support from the Na-tional Key Research and Development Program of China(2017YFA0505103); Research Fund for the Doctoral Pro-gram of Higher Education of China 20120002110022 andNational Natural Science Foundation of China (31471254)to J.D. This work was also supported by the NationalInstitutes of Health (NIH) grant U54GM103520 to J.D.B.

Note added in proof: See Jiang et al. 2017 (pp. 3857–3866) in G3: GenesjGenomesjGenetics for a related work.

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Communicating editor: O. Rando

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